Nuclei pure prep nuclei isolation kit

Nuclei were isolated from iPSNs and postmortem human motor and occipital cortex tissue using the Nuclei Pure Prep Nuclei Isolation Kit (Sigma Aldrich) following manufacturer protocol with slight modifications as previously described [4 , 5 (link)]. About 10 million iPSNs or 100 mg of frozen postmortem motor cortex tissue (obtained from the Target ALS Human Postmortem Tissue Core (see Additional file 2: Table 2 for demographic information) was used for nuclei isolation. A 1.85 M sucrose gradient was used to enrich for neuronal nuclei. Following isolation, nuclei were centrifuged onto collagen coated (1 mg/mL; Advanced Biomatrix) slides with a CytoSpin 4 centrifuge (Thermo Fisher Scientific) and immunostained as previously described [4 , 5 (link)] (see Additional file 2: Table 3 for antibody information). Isolated nuclei were subsequently imaged by super resolution structured illumination microscopy (SIM) using a Zeiss ELYRA S1 as previously described [4 , 5 (link)]. All images were acquired using identical imaging parameters (e.g. laser power, gain) and subjected to default SIM deconvolution and processing in Zeiss Zen Black 2.3 SP1. Representative images are presented as 3D maximum intensity projections generated in Zeiss Zen Black 2.3 SP1. Images were faux colored green for contrast and display.

The protocol to obtain single nuclei RNA-sequencing data and initial pre-processing steps were described in Chapter 3. To summarize briefly, nuclei were isolated from fresh frozen tissue samples following the Nuclei Pure Prep nuclei isolation kit (Sigma-Aldrich, St. Louis, MO). Each sample was multiplexed with lipid-tagged oligonucleotides following the MULTI-seq protocol^{138 (link)}. Libraries for single nuclei RNA-seq were prepared following the 10X Genomics Single Cell Gene Expression workflows (10X Genomics, Pleasanton, CA). Libraries were pooled and sequenced using the lllumina NextSeq500 instrument. 10X Cell Ranger software was used to align sequences to the GRCh38 pre-mRNA reference genome.
Low-quality nuclei, as defined as having greater than 10,000 and less than 2,000 features and more than 5% of reads that map to mitochondrial genes, were removed for analyses. Samples were demultiplexed using an integrative approach, combining barcode based demultiplexing and genotype-based demultiplex method^{139 (link),140 (link)}. Downstream analyses for single nuclei-RNA seq were done with the Seurat package v4 in R^{139 (link),141 (link)–143 (link)}.

Nuclei of cells were isolated using the Nuclei Pure Prep Nuclei Isolation kit (Sigma, NUC201) following the instruction of the manufacturer. The cytoplasmic or nuclei fractions could be separated and nuclei fractions were used for metabolite detection and immunofluorescence. The content of metabolites including pyruvate, citrate, α-KG, and acetyl-CoA was detected immediately after nuclei isolation. For the immunostaining of the TCA cycle enzymes specifically in the nucleus, isolated nuclei were dropped on the coverslips and fixed with 4% PFA immediately after drying and stained according to the above immunofluorescence protocol. The kits used are listed in Supplementary Table 3.

A detailed description of the heart cell isolation procedure for the tabula muris can be found in their supplementary information of the online version under “A detailed discussion of organ cell types” [17 (link)].
Single nuclei were isolated as previously described [11 (link)]. In brief, whole hearts were harvested whereby the pulmonary artery and vena cava were removed as far as possible. The hearts were then pooled for each strain before using the Nuclei PURE Prep nuclei isolation kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. For this, tissue samples were rinsed with ice-cold PBS and minced thoroughly with a scalpel. Remaining blood was removed by rinsing with PBS before the tissue chunks were further homogenized in chilled lysis solution using a gentle MACS dissociator. Lysate samples were mixed with chilled sucrose cushion solution and layered on 1.8 M sucrose cushion solution for density gradient centrifugation for 45 min at 30.000× g and 4 °C. Nuclei pellets were resuspended in chilled PBS containing 1% BSA and 0.2 U/µL RNase inhibitor and cell debris were removed by using 40 µm Flowmi cell strainers. After another centrifugation for 8 min at 600× g and 4 °C, nuclei were resuspended in Nuclei PURE storage buffer, snap-frozen in liquid nitrogen, and stored at −80 °C until processing.

Nuclei were extracted from 1x10⁷-10⁸ cells/well according to the Nuclei Pure Prep Nuclei Isolation Kit protocol (Sigma-Aldrich NUC-201). Samples were layered on a 1.8M sucrose cushion concentration (30 ml per 10 ml homogenized samples) and spun at 30000g for 45 min at 4°C (Beckman Optima L-90K Ultracentrifuge, SW32 rotor). Supernatant were discarded and pellet were resuspended in 200 μl Nuclei PURE storage buffer [5 (link)]. MicroDNA was isolated using the Qiagen High Speed Midi Plasmid Purification Kit according to the manufacturer instructions. DNA was eluted in 1 ml of Tris-EDTA (TE) buffer solution and concentrated by adding 20 μg of glycogen, 0.1 volume of 3M sodium acetate, pH 5.2 and 2 volume of Isopropanol followed by centrifugation at 16000g for 20 minutes. The pellet was resuspended in 20 μl 1X TE and concentration determined using the Nanodrop. 1 μg was digested with the Exonuclease VII (Epicentre) and an ATP-Dependant DNase (Epicentre) to remove linear ds and ssDNA, and purified after each digestion step with MinElute.